TOYOBO Ideas & Chemistry Biotechnology Operating
Department

HXK-311

PREPARATION and SPECIFICATION

Appearance White amorphous powder, lyophilized
Activity GradeⅢ　150 U/mg-solid or more
Contaminants Phosphoglucose isomerase ≤1.0×10-1 %
6-Phosphogluconate dehydrogenase ≤1.0×10-2 %
Glucose-6-phosphate dehydrogenase ≤1.0×10-2 %
Myokinase ≤1.0×10-2 %
Glutathione reductase ≤5.0×10-1%

PROPERTIES

Stability Stable at −20 ℃ for at least one year(Fig.1)
Molecular weight approx. 82,000 (by gel filtration)
Isoelectric point 4.1±0.1
Michaelis constants 2.3×10-4 M (D-Glucose), 7.7×10-5 M (ATP)
Inhibitors Metal ions, p-chloromercuribenzoate, iodoacetamide, SDS, etc
Optimum pH 8.0−9.0(Fig.2)
Optimum temperature 50 ℃(Fig.3)
pH Stability pH 4.0−9.0 (25 ℃, 20 hr)(Fig.4)
Thermal stability below 45 ℃ (pH 7.0, 30 min)(Fig.5)
Substrate specificity (Table 1)
Effect of various chemicals (Table 2)

APPLICATIONS

The enzyme is useful for enzymatic determination of glucose, adenosine-5'-triphosphate (ATP) and creatine phosphokinase in combination with glucose-6-phosphate dehydrogenase (=G-6-PDH, G6D311, G6D-321).

ASSAY

Principle

Principle

Principle

The formation of NADH is measured at 340 nm by spectrophotometry.

Unit definition

One unit causes the formation of one micromole of NADH per minute under the conditions detailed below.

Method

Reagents

A. Tris-HCl buffer, pH 8.0 50 mM, containing 13.3 mM MgCl2
B. Glucose solution 0.67 M in Tris-HCl buffer solution (A) (The solution should be keep at room temperature for at least 1 hour before use)
C. ATP solution 16.5 mM in Tris-HCl buffer solution (A) (Should be freshly prepared)
D. NAD＋ solution 6.8 mM in Tris-HCl buffer solution (A) (Should be freshly prepared)
E. G-6-PDH solution 300 U/mL (Dilute with Tris-HCl buffer solution (A) and store on ice)
F. Enzyme diluent Tris-HCl buffer solution (A) containing 0.1 % of bovine serum albumin

Procedure

1. Prepare the following reaction mixture in a cuvette (d＝ 1.0cm) and equilibrate at 30℃ for approximately 5 minutes.

2.30 mL Tris-HCl buffer solution (A)
0.50 mL Glucose solution (B)
0.10 mL ATP solution (C)
0.10 mL NAD＋ solution (D)
0.01 mL G-6-PDH solution (E)
Concentration in assay mixture
Tris-HCl buffer 50 mM
Glucose 0.11 M
ATP 0.53 mM
NAD＋ 0.22 mM
MgCl2 13 mM
BSA 3.2 μg/mL
G-6-PDH approx.1.0 U/mL

2. Add 0.1ml of the enzyme solution* and mix by gentle inversion.

3. Record the increase of optical density at 340 nm against water for 4 to 5 minutes with a spectrophotometer thermostated at 30 ℃ and calculate the ΔOD per minute from the initial portion of the curve (ΔOD test).

At the same time, measure the blank rate (ΔOD blank) by the same method as the test except that the enzyme diluent (F) is added instead of the enzyme solution.

*Dissolve the enzyme preparation on ice-cold enzyme diluent (F) and dilute to 0.1−0.3 U/mL with the same buffer, immediately before the assay.

Calculation

Activity can be calculated by using the following formula :

  • Volume activity (U/ml) =

  • ΔOD/min (OD test−OD blank)×Vt×df

    6.22×1.0×Vs

  • ＝ ΔOD/min×5.0×df

Weight activity (U/mg) ＝ (U/ml)×1/C

Vt : Total volume (3.11 mL)
Vs : Sample volume (0.1 mL)
1.0 : Light path length (cm)
6.22 : Millimolar extinction coefficient of NADH (cm2/micromole)
df : Dilution factor
C : Enzyme concentration in dissolution (c mg/mL)

Table 1. Substrate specificity of Hexokinase

[Pyruvate kinase-Lactate dehydrogenase system with 0.1 M Tris-HCl buffer, pH 7.5]

  • Substrate(100mM) Relative activity(%)
    D-Glucose 100
    D-Fructose 140
    D-Mannose 52
    2-Deoxy-D-glucose 91
  • Substrate(100mM) Relative activity(%)
    D-Galactose 0
    D-Xylose 2
    D-Glucosamine 58

Table 2. Effect of Various Chemicals on Hexokinase

[The enzyme dissolved in 50mM K-phosphate buffer, pH 6.5 (5 U/mL) contg. 0.1 % bovine serum albumin was incubated with each chemical at 30 ℃ for 1hr.]

  • Chemical Concn.(mM) Residual
    activity(%)
    None - 100
    Metal salt
    AgNO3 2.0 0
    BaCl2 2.0 99
    CaCl2 2.0 98
    CdCl2 2.0 85
    CoCl2 2.0 85
    CuSO4 2.0 25
    FeCl3 2.0 28
    FeSO4 2.0 80
    HgCl2 2.0 0
    MgCl2 2.0 98
    MnCl2 2.0 100
    NiCl2 2.0 100
    Pb(OAc)2 2.0 98
    Zn(OAc)2 2.0 98
    ZnSO4 2.0 99
    NaF 20.0 101
    NaN3 20.0 102
  • Chemical Concn.(mM) Residual
    activity(%)
    PCMB 2.0 0
    MIA 2.0 80
    IAA 2.0 7
    EDTA 5.0 103
    (NH4)2SO4 20.0 104
    Borate 20.0 102
    o-Phenanthroline 2.0 101
    α,α′-Dipyridyl 2.0 102
    Urea 2.0 104
    Guanidine 2.0 103
    Hydroxylamine 2.0 104
    Na-cholate 1.0 % 102
    Triton X-100 1.0 % 105
    Brij 35 1.0 % 0
    SDS 0.1 % 25
    Tween 20 0.1 % 101
    Span 20 0.1 % 106
    DAC 0.1 % 101

Ac, CH3CO; NEM, N-Ethylmaleimide; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; IAA, Iodoacetamide; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride.

  • Fig.1. Stability (Powder form)

    Fig.1. Stability (Powder form)

    (kept under dry conditions)

  • Fig.2. pH-Activity

    Fig.2. pH-Activity

    30 ℃ in the 50 mM buffer solution: pH 6.2-7.5, PIPES-NaOH: pH 7.5-9.0, Tris-HCI: pH 9.0-10.0, Glycine-NaOH

  • Fig.3. Temperature activity

    Fig.3. Temperature activity

    (in 50 mM Tris-HCI buffer,pH 8.0)

  • Fig.4. pH-Stability

    Fig.4. pH-Stability

    25 ℃, 20 hr-treatment in the 0.1 M buffer solution: pH 4.0-8.0,Acetate-NaOH;pH 6.0-8.0, K-phosphate; pH 7.5-9.0,Tris-HCl;pH 9.0-10.5, Glycine-NaOH enzyme concn.: ca.10 U/mL

  • Fig.5. Thermal stability

    Fig.5. Thermal stability

    30 min-treatment with 50 mM K-phosphate buffer, pH 7.0, containing 0.1% bovine serum albumin enzyme concn.: ca.5 U/mL

活性測定法（Japanese）

1. 原理

原理

原理

NADHの生成量を340nmの吸光度の変化で測定する。

2.定義

下記条件下で1分間に1マイクロモルのNADHを生成する酵素量を1単位(U)とする。

3.試薬

  • 50mM Tris-HCl緩衝液,pH8.0 (13.3mM MgCl2を含む)
  • 0.67Mグルコース緩衝液(試薬Aで溶解)(溶解後,室温に少くとも1時間保管したものを使用する)
  • 16.5mM ATP溶液(試薬Aで溶解)(用時調製)
  • 6.8mM NAD＋溶液(試薬Aで溶解)(用時調製)
  • G-6-PDH溶液(300U/mgに試薬Aで希釈し氷冷保存する)

酵素溶液：酵素標品を予め氷冷した0.1%牛血清アルブミン(BSA)を含む試薬Aの緩衝液で溶解し，同溶液で0.1〜0.3U/mgに希釈して氷冷保存する。

4.手順

1.下記反応混液をキュベット(d＝1.0cm)に調製し,30℃で約5分間予備加温する。

2.30ml Tris-HCl緩衝液 (A)
0.50ml グルコース溶液 (B)
0.10ml ATP溶液 (C)
0.10ml NAD＋水溶液 (D)
0.01ml G-6-PDH溶液 (E)

2.酵素溶液を0.1mgを添加し,ゆるやかに混和後,水を対照に30℃に制御された分光光度計で,340nmの吸光度変化を4〜5分間記録し,その初期直線部分から1分間当りの吸光度変化を求める(ΔOD test)。

3.盲検は反応混液①に酵素溶液の代りに酵素希釈液(0.1%BSAを含む試薬A)を0.1mg加え,上記同様に操作を行って1分間当りの吸光度変化を求める。(ΔODblank)。

5.計算式

  • U/ml =

  • ΔOD/min (OD test−OD blank)×3.11(mg)×希釈倍率

    6.22×1.0×0.1(mg)

＝ ΔOD/min×5.0×希釈倍率
U/mg = U/ml×1/C
6.22 : NADHのミリモル分子吸光係数(cm2/micromole)
1.0 : 光路長(cm)
C : 溶解時の酵素濃度(c mg/ml)